Missense mutation causes multiple defects in Nav1.4 channel gating and leads to an SCN4A-associated overlap phenotype

Mutations in SCN4A gene encoding the skeletal muscle-type voltage-gated sodium channel Nav1.4 are known to cause neuromuscular disorders. Here, we report a previously undescribed variant affecting the DIII-IV cytoplasmic linker, which is a critically important region participating in channel inactivation. The variant p.L1326P was found in heterozygous state in two members of the same family presenting mild symptoms of periodic paralysis and in vivo electrophysiological features of sodium channel myotonia. Functional characterization was performed using Xenopus oocytes co-expressing human Nav1.4 and β1 subunits, and the two-electrode voltage clamp technique. We recorded gating alterations in Nav1.4-L1326P compared with wild-type channels. (1) The voltage dependence of activation shifted to the depolarizing direction, whereas (2) the voltage dependence of steady-state inactivation moved in the hyperpolarizing direction. (3) Fast channel inactivation was markedly decelerated in Nav1.4-L1326P. In addition, (4) we observed a dramatic increase in persistent current. The gain-of-function effects are consistent with the consensus view on the pathogenesis of myotonia and periodic paralysis. AlphaFold 3 models suggest stronger contacts between the cytoplasmic domain and the DIII-IV linker in the mutant channels offering a possible structural interpretation of the functional changes.